Production of cellulase EglA by recombinant Bacillus megaterium

• Main Authors: Chia-En Wu, 吳佳恩
• Other Authors: 黃慶璨
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/67317028968637474232

碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 93 === Cellulose, the major component of plant cell walls, is the most abundant renewable biomass in nature and a sustainable clean resource for energy. Cellulase is responsible for the first step of degradation of cellulose. The application potential of cellulase includes food, brewery and wine, animal feed, textile and laundry, pulp and paper, as well as agricultures. Several homologous or heterologous expression systems, such as bacteria, yeasts or filamentous fungi, have been developed for production of cellulase. Bacillus megaterium has several advantages over E. coli for a broad industrial use in the production of recombinant proteins. First, ease of culture, non-pathogenic and no inclusion body. Second, there is no alkaline proteases. Last, the recombinant plasmids in B. megaterium is stable structurally and segregationally. In this study, we described the establishment of a B. megaterium based recombinant protein production and export system for the cellulase EglA from Piromyces rhizinflatus. We modified the B. megaterium by adapting E. coli T7 system for over-production of proteins and inserting a signal peptide from homologous β-amylase gene for protein secretion. The goal of this study is to develop an alternative expression system which could secret the desired protein into the culture supernatant. We have constructed secretory cloning vector pWH1520-S, xylose-inducible T7 RNA polymerase vector pWH1520-T7R, expression plasmid pWH1520-C. We also cloned T7 promoter gene and inserted eglA gene downstream. We have successfully express cellulase EglA in B. megateirum WH1520-C, and 5 Units/mL of crude extraction were measured.